1. Field of the Invention
This invention relates to a method for producing protein, and more specifically this invention relates to a method for expressing and isolating heterologous proteins using photosynthetic organisms.
2. Background of the Invention
Proteins are routinely divided into soluble proteins and membrane proteins. Membrane proteins are extremely important for normal cell function. They provide the means by which cells communicate, transduce signals and transport metabolites between internal compartments, and build gradients of ions which are used to fuel all ingrained activities. Membrane proteins are one of the early defenses against invading foreign organisms.
Although roughly 35% of the proteins known or expected to be found in most organisms are membrane-associated, little structural or functional information exists on these proteins relative to their soluble peers. Any new information on membrane protein structures would aid biologists, physicists and chemists in their understanding of important structural relationships necessary for essential protein functions in lipid bilayer environments. Using traditional methodology, it has been difficult to purify quantities of native membrane-associated proteins that are sufficient for experiments. Inasmuch as the functional properties and stability of membrane proteins are dependent upon the lipid micelle surrounding them, these proteins often denature or otherwise deviate from their native states when removed from their natural environs. Additionally, most membrane proteins are most commonly expressed at very low levels, in amounts insufficient for purification and crystallization. To date, the three dimensional structures of only a dozen unique membrane proteins are known, in comparison to the structures of approximately 1500 soluble proteins.
Knowledge of the structures and a determination of the function of membrane proteins would contribute greatly to our understanding of biological processes. For example, in recent years, structure-based rational drug design has produced powerful competitive inhibitors of cofactor binding in enzyme catalysis. Because of their importance in cellular functions that can contribute to various disease states, membrane proteins are targets for drug discovery that impacts disease control and prevention.
Purification of membrane proteins from their native host cells has been attempted by removing the protein from its native (hydrophobic) surroundings and placing same in small detergent micelles which attempt to mimic the lipid environment. Following this solubilization process, routine chromatography or precipitation techniques (which have been perfected for soluble proteins) are utilized to purify and crystallize the solubilized membrane proteins. Such adaptations rarely yield large amounts of the membrane protein in functional form.
Efforts have been made to create a process whereby membrane-associated proteins are over-expressed and subsequently purified from native hosts or host cells of another organism (i.e., heterologous expression). To some degree, these efforts have all utilized a combination of a desired coding sequence with a foreign promoter known to induce high levels of protein synthesis. For example, U.S. Pat. No. 5,310,663 (Dobeli et al.) describes fusion proteins, comprising a coding sequence of a desired protein and the coding gene sequence of an affinity peptide, wherein the affinity peptide is attached and used to purify the desired protein product. Purification is accomplished using metal chelate affinity chromatography in nitrilotriacetic acid resins. However, no provision exists for circumventing the unique and inherent difficulties associated with purifying intact hydrophobic proteins.
U.S. Pat. No. 5,750,374 (Dobeli et al.) provides a process for producing hydrophobic polypeptides and proteins by using the fusion protein technology of the prior ""663 patent. Like the ""663 patent, this process links a coding gene sequence of a desired protein with the coding sequence of an affinity peptide to create a fusion protein. This process provides an additional means of purifying the desired protein through chemical or enzymatic cleavage at a strategic cleavage site. However, the ""374 patent has no provision for maintaining the intact, tertiary and quaternary structure of the desired hydrophobic protein.
A method for the heterologous overexpression of hydrophobic proteins has been disclosed by Turner et al. Protein Expression and Purification, 17, pp. 312-323 (1999). Coding regions of desired membrane proteins are juxtaposed with the bacterio-opsin (bop) regulatory sequences in the cell membrane of Halobacterium salinarum. While attempts at overexpression have been successful, this process does not provide for simultaneous production and sequestration or compartmentalization of the desired protein.
R. van Dijk et al. Enzyme and Microbial Technology 26 9-10, pp 793-800 describe a heterologous overexpression system based on Hansenula polymorpha. This method suggests the utilization of peroxisomes in which produced proteins may accumulate. However, as with the H. salinarum system discussed supra, no provision exists for the simultaneous production and compartmentalization of the targeted components, inasmuch as the promoters utilized there in are for the most part constitutive.
A need exists in the art for a generalized system for the heterologous expression and recovery of functional membrane proteins. Ideally, the system should incorporate a means to control inducement of expression of the proteins which is simultaneous with the sequestration and therefore isolation of the protein. The system also should utilize simple purification protocols.
It is an object of the present invention to provide a method for heterologous expression of membrane proteins that overcomes many of the disadvantages of the prior art.
It is another object of the present invention to provide a method for purifying proteins. A feature of this method is that synthesis of the protein occurs at the same time a membrane to encapsulate that protein is generated. An advantage of this method is that the generated protein is sequestered in a compartment in its native state while it is produced.
Still another object of the present invention is to utilize a photosynthetic system to produce transmembrane proteins. A feature of this invented method is the utilization of a promoter, which responds to the same environmental cues as do promoters for membrane synthesis, to produce the transmembrane proteins. For example, the invented process utilizes a promoter for proteins of the photosynthetic membrane of Rhodobacter to also facilitate production of the transmembrane protein. An advantage of this method is that well-known xe2x80x9cindicatorsxe2x80x9d such as color changes in the Rhodobacter system can be used as a monitor of culture conditions that favor the synthesis of heterologous proteins from the inducible promoter.
Yet another object of the present invention is to provide a method for purifying transmembrane proteins. A feature of this invented method is to append an affinity tag to the protein. An advantage of the invented method is that the tag facilitates simple, rapid, and less disruptive extraction of the formed protein from its native membrane environment so that the protein retains its structural and functional integrity for further study.
A further object of the present invention is to provide a method to facilitate the controlled over-expression of heterologous membrane proteins. A feature of the method is that intracytoplasmic membrane (ICM) used to harbor the expressed foreign membrane proteins is both inducible and readily isolated. The extent of induction, and thus the amount of foreign protein that is synthesized, can be adjusted continuously. An advantage of the method is that this parallel induction of both the foreign membrane protein and its preferred environment will favor insertion of the expressed membrane protein into newly developing ICM, thereby decreasing the chances of inclusion body formation. Another advantage is that the same culture manipulations can be used at the same time to introduce heavy metal, scattering moieties into the gene products to facilitate subsequent structure determination with multiple-wavelength anomalous dispersion (MAD) techniques.
Briefly, the invention provides a method for production of heterologous proteins comprising simultaneous expression of both the protein and a hospitable environment which facilitates in situ sequestration and therefore rudimentary in vivo purification of the protein.
The invention also provides a method for simultaneously producing and sequestering proteins, including functional membrane proteins.
The invention further provides a method for simultaneously producing and compartmentalizing a fusion protein, the method comprising selecting a photosynthetic organism having promoters for producing a membrane compartment; placing expression of the fusion protein under the control of one of these promoters; and activating that promoter.
Also provided is a DNA sequence that will transcribe as mRNA, the sequence comprising a puf-promoted or a puc-promoted gene that will result in a stable transcript and the translation of biologically active polypeptides linked to an affinity peptide, and that will also result in the simultaneous isolation/purification of the polypeptides in their native state.
Further provided are DNA sequences that will transcribe as mRNA comprising an RNA stem-loop stabilizing region; and a transcript attached to said region that will result in the translation of biologically active polypeptide linked to an affinity peptide, and that will result in the simultaneous compartmentalization of the polypeptide in its native state.
Also provided is a method to identify products of genes which interact to form stable, multi-subunit membrane-associated protein complexes.